Recovery of structurally intact and infectious poliovirus type 1 from HeLa cells during Receptor-mediated endocytosis

113

Poliovirus (PV), when injected intramuscularly into the calf, is incorporated into the sciatic nerve and causes an initial paralysis of the inoculated limb in transgenic mice carrying the human PV receptor (hPVR/CD155) gene. Here, we demonstrated by using an immunoelectron microscope that PV particles exist on vesicle structures in nerve terminals of neuromuscular junctions. We also demonstrated in glutathione S-transferase pulldown experiments that the dynein light chain, Tctex-1, interacts directly with the cytoplasmic domain of hPVR. In the axons of differentiated rat PC12 cells transfected with expression vectors for hPVRs, vesicles composed of PV and hPVR␣, as well as a mutant hPVR␣ (hPVRM␣) that had a reduced ability to bind Tctex-1, colocalized with Tctex-1. However, vesicles containing PV, dextran, and hPVR␣ had only retrograde motion, while those containing PV, dextran, and hPVRM␣ had anterograde or retrograde motion. Topical application of the antimicrotubule agent vinblastine to the sciatic nerve reduced the amount of virus transported from the calf to the spinal cord. These results suggest that direct efficient interaction between the cytoplasmic domain and Tctex-1 is essential for the efficient retrograde transport of PV-containing vesicles along microtubules in vivo.In humans, paralytic poliomyelitis is considered to result from an invasion by circulating poliovirus (PV) into the central nervous system, probably via the blood-brain barrier. The notion is supported by a previous study using a mouse model (44). In that study, it was demonstrated that circulating PV after intravenous inoculation appears to cross the blood-brain barrier at a high rate, and the neural dissemination pathway from the skeletal muscle is not the primary dissemination route of the circulating virus to the central nervous system. Along with this pathway of dissemination, a neural pathway has been reported in humans (32), monkeys (20), and PV-sensitive transgenic (Tg) mice carrying the human PV receptor (hPVR/ CD155) gene (35,36), and it appears to be important in causing provocation poliomyelitis (14). Using the Tg mouse line, we demonstrate that PV inoculated into the calf is incorporated into the sciatic nerve and retrogradely transported through the axons as intact virion particles, that one of the fast retrograde axonal transport systems is involved in viral dissemination, and that the pathogenesis of PV infection via the neural pathway is inhibited by the anti-hPVR monoclonal antibody (MAb) p286, which is able to block the infection (35).hPVR is a member of the immunoglobulin (Ig) superfamily, with three linked extracellular Ig-like domains followed by a membrane-spanning domain and a cytoplasmic domain (CP). Two membrane-bound forms (hPVR␣ and hPVR␦) and two secreted forms (hPVR␤ and hPVR␥) derived by alternative splicing are potentially expressed in human cells (28). Membrane-bound hPVRs are considered to play important roles in the early steps of infection, such as binding of the virus to the cell surface, penetration...

Section: On May 10 2018 By Guestmentioning

confidence: 71%

“…10B) (43). However, DeTulleo and Kirchhausen (10) found by using a dominantnegative mutant of dynamin that PV might not require the clathrin pathway.…”

Section: Discussionmentioning

confidence: 99%

Receptor (CD155)-Dependent Endocytosis of Poliovirus and Retrograde Axonal Transport of the Endosome

Ohka

Matsuda

Tohyama

et al. 2004

113

“…Another important human picornavirus, poliovirus, may also enter the cell via endosomes; however, it is likely that poliovirus uses multiple pathways for infection (6). In HeLa cells, virus particles have been visualized inside endosome-like structures at early times postinfection, suggesting that poliovirus infection requires virus uptake into endosomes (49,82). This route of infection has also been reported for Hep-2 cells (84) and neural cells (59).…”

mentioning

confidence: 82%

Early Events in Integrin αvβ6-Mediated Cell Entry of Foot-and-Mouth Disease Virus

Berryman¹,

Clark²,

Monaghan

et al. 2005

108

103

We have shown that foot-and-mouth disease virus (FMDV) infection mediated by the integrin ␣v␤6 takes place through clathrin-dependent endocytosis but not caveolae or other endocytic pathways that depend on lipid rafts. Inhibition of clathrin-dependent endocytosis by sucrose treatment or expression of a dominantnegative version of AP180 inhibited virus entry and infection. Similarly, inhibition of endosomal acidification inhibited an early step in infection. Blocking endosomal acidification did not interfere with surface expression of ␣v␤6, virus binding to the cells, uptake of the virus into endosomes, or cytoplasmic virus replication, suggesting that the low pH within endosomes is a prerequisite for delivery of viral RNA into the cytosol. Using immunofluorescence confocal microscopy, FMDV colocalized with ␣v␤6 at the cell surface but not with the B subunit of cholera toxin, a marker for lipid rafts. At 37°C, virus was rapidly taken up into the cells and colocalized with markers for early and recycling endosomes but not with a marker for lysosomes, suggesting that infection occurs from within the early or recycling endosomal compartments. This conclusion was supported by the observation that FMDV infection is not inhibited by nocodazole, a reagent that inhibits vesicular trafficking between early and late endosomes (and hence trafficking to lysosomes). The integrin ␣v␤6 was also seen to accumulate in early and recycling endosomes on virus entry, suggesting that the integrin serves not only as an attachment receptor but also to deliver the virus to the acidic endosomes. These findings are all consistent with FMDV infection proceeding via clathrin-dependent endocytosis.

“…For picornaviruses, contradictory data about the entry mechanisms have been reported. For example, polioviruses have been suggested to infect cells through directly forming pores in the cell membrane (40), by clathrin-coated endocytosis (46,47), or by crossing a membrane barrier in the form of a 135S particle leading to the further uncoating in the cytoplasm (19). Recently, DeTulleo and Kirchhausen compared the entry mechanisms of human rhinovirus 14 (HRV14) and poliovirus 1 in HeLa cells and, based on studies with mutated dynamin, suggested that HRV14 utilizes a clathrin-mediated endocytosis pathway, whereas poliovirus 1 does not appear to use this route (9).…”

Section: Discussionmentioning

confidence: 99%

Internalization of Echovirus 1 in Caveolae

Marjomäki

Pietiäinen

Matilainen

et al. 2002

203

211

Echovirus 1 (EV1) is a human pathogen which belongs to the Picornaviridae family of RNA viruses. We have analyzed the early events of infection after EV1 binding to its receptor ␣2␤1 integrin and elucidated the route by which EV1 gains access to the host cell. EV1 binding onto the cell surface and subsequent entry resulted in conformational changes of the viral capsid as demonstrated by sucrose gradient sedimentation analysis. After 15 min to 2 h postinfection (p.i.) EV1 capsid proteins were seen in vesicular structures that were negative for markers of the clathrin-dependent endocytic pathway. In contrast, immunofluorescence confocal microscopy showed that EV1, ␣2␤1 integrin, and caveolin-1 were internalized together in vesicular structures to the perinuclear area. Electron microscopy showed the presence of EV1 particles inside caveolae. Furthermore, infective EV1 could be isolated with anti-caveolin-1 beads 15 min p.i., confirming a close association with caveolin-1. Finally, the expression of dominant negative caveolin in cells markedly inhibited EV1 infection, indicating the importance of caveolae for the viral replication cycle of EV1.